This invention relates to retroviral vectors and their use in methods of mammalian gene mutagenesis.
Eukaryotic genomes are estimated to contain 6,000-80,000 genes (Collins, Proc. Natl. Acad. Sci. USA 92:10821-10823 (1995)). Even in the best characterized organisms, the function of the majority of these genes is unknown. In addition, relatively little information is available concerning the fraction of the genome that is expressed in particular cell types or the cellular processes in which specific gene products participate. In an attempt to decipher genes' functions, large scale mutagenesis screens have been developed and have proven instrumental in unraveling the roles of certain genes in organisms such as Drosophila melanogaster (Nusslein-Volhard and Wieschaus, Nature 287:795-801 (1980); Ballinger and Benzer, Proc. Natl. Acad. Sci. USA 86:9402-9406 (1989); Kaiser and Goodwin, Proc. Natl. Acad. Sci. USA 87:1686-1690 (1990); and Spradling et al., Proc. Natl. Acad. Sci. USA 92:10824-10830 (1995)), Caenorhabditis elegans (Hirsh and Vanderslice, Dev Biol. 49:220-235 (1976); and Zwaal et al., Proc. Natl. Acad. Sci. USA 90:7431-7435 (1993)), Zebrafish (Solnica-Krezel et al., Genetics 136:1401-1420 (1994); and Riley and Grunwald, Proc. Natl. Acad. Sci. USA 92:5997-6001 (1995)), Arabidopsis (Jurgens et al., Development Suppl. 1:27-38 (1991); Mayer et al., Nature 353:402-407 (1991); and Sundaresan et al., Genes Dev. 9:1797-1810 (1995)), Maize (Scanlon et al., Genetics 136:281-294 (1994); and Osborne and Baker, Curr. Opin. Cell Biol. 7:406-413 (1995)), and Saccharomyces cerevisiae (Burns et al., Genes Dev. 8: 1087-1105 (1994); and Chun and Goebl, Genetics 142:30-50 (1996)). In mammals, however, these approaches have generally been limited by the large genome size and the development of the embryo inside a mother's uterus.
Some progress has been made in understanding mammalian gene function as a result of the development of mouse embryonic stem (ES) cell technology. This technology has significantly altered the field of mammalian genetics by allowing the bulk of genetic manipulations to be executed in vitro (Evans and Kaufman, Nature 292:154-156 (1981); Bradley et al., Nature 309:255-256 (1984); and Robertson, Trends Genet. 2:9-13 (1986)). This is possible because mouse ES cells are pluripotent, that is, they have the ability to generate entirely ES cell-derived animals. Accordingly, gene inactivation in mouse ES cells and subsequent generation of "knock-out" (KO) mice is a powerful method for gaining information about the function of a gene in a whole animal system. If desired, genetic alterations, such as gene KOs which inactivate genes, may be introduced into these cells, and their consequences may be studied in the whole animal (Jaenisch, Science 240:1468-1474 (1988); and Rossant and Nagy, Nat. Med. 1:592-594 (1995)).
Currently, the available mouse mutagenesis methodologies are somewhat limited in their general utility as gene function screening systems. Gene targeting, the most widely used approach, is laborious and time consuming (Capecchi, Science 244:1288-1292 (1989)). And gene trap and chemical/radiation induced mutagenesis are generally restricted in their targets (Gossler et al., Science 244:463-465 (1989); Friedrich and Soriano, Genes Dev. 5:1513-1523 (1991); Skarnes et al., Genes Dev. 6:903-918 (1992); von Melchner et al., Genes Dev. 6:919-927 (1992); Reddy et al., Proc. Natl. Acad. Sci. USA 89:6721-6725 (1992); Takeuchi et al., Genes Dev. 9:1211-1222 (1995); and Takahashi et al., Science 264:1724-1733 (1994)). The gene trap approach is limited to genes expressed in ES cells, although variations of the method have been developed for targeting specific subclasses of genes expressed in early embryonic stages (Wurst et al., Genetics 139:889-899 (1995); Skames et al., Proc. Natl. Acad. Sci. USA 92:6592-6596 (1995); and Forrester et al., Proc. Natl. Acad. Sci. USA 93:1677-1682 (1996)). And the chemical/radiation induced mutagenesis technique is generally limited to genes that can result in dominant phenotypes when mutated. None of these approaches, as currently exploited, may be readily streamlined or automated, nor can they be readily adapted to carry out saturated mutagenesis of the mouse genome.